N-demethylcelesticetin derivatives

ABSTRACT

NEW ANTIBIOTICS, N-DEMETHYL-7-O-DEMETHYLCELESTICETIN (IV) AND N-DEMETHYLCELESTICETIN (IVA), ARE PRODUCED CONCOMITANTLY BY THE CONTROLLED FERMENTATION OF THE NEW MICRO-ORGANISM STREPTOMYCES CALESTIS STRAIN 22218A IN AN AQUEOUS NUTRIENT MEDIUM. THESE ANTIBIOTICS ARE ANTIBACTERIALLY ACTIVE AND ALSO CAN BE CONVERTED TO VARIOUS ANTIBACTERIALLY ACTIVE ANALOGUES.

Unimd Stew Pa nt 3 812 096 N-DEMETHYLCELliSTIbETlN DERIVATIVES Alexander D. Argoudelis, Portage, and John H. Coats and Oldrich K. Sebek, Kalamazoo, Mich., assignors to The Upjohn Company, Kalamazoo, Mich. No Drawing. Filed June 12, 1972, Ser. No. 261,724

Int. Cl. C07c 129/18 US. Cl. 260--210 R 4 Claims H 4 1 0 K011 H a 8 a \l3 2 SCHiCHaO H OH I Celesticetin can be hydrolyzed according to the process disclosed in US. Pat. 2,851,463 to produce the compound desalicetin which can be represented by the following 40 structuralformula:

K H J These celesticetin'compounds are structurally related somewhat to the well-known antibiotic lincomycin which can be represented by the following structural formula:

HO J; H K011 11X EL CH:

BRIEF SUMMARY OF THE INVENTION The novel compounds of the invention, N-demethyl-7- O-demethylcelesticetin (U-39,579) and N-demethylcelesticetin (U40,585), are obtained by culturing Streptomyces caelestis strain 2221821, NRRL 5481, in an aqueous Patented May 21, 1974 ice nutrient medium. N-demethyl-7 O demethylcelesticetin can be represented by the following structural formula:

I on, no-bn H b v E-NIH 0 5 HO H H OH H) i I I somomoQ H OH IV N-demethylcelesticetin can be represented by the following structural formula:

II I

C CHgO-AlH H in;

g K l t OH H CHgCHzOE Upon the treatment of N-demethyl-7-O-demethylcelesticetin (IV) with a suitable base, for example, sodium hydroxide, potassium hydroxide, ammonium hydroxide, and the like, there is produced N-demethyl-7-O-demethyldesalicetin which can be represented by the followin structural formula:

OH v

Likewise, upon treatment of N-demethylcelesticetin (IVa) with a base, as above, there is produced N-demethyldesalicetin which can be represented by the following structural formula:

H cm

cmo-b Treatment of N-demethy1-7-O-demethylcelesticetin with hydrazine hydrate yields B-hydroxyethylthiolincosaminide (B-HETL) which can be represented by the following structural formula:

H: HO-JJH OH H 1![ SCHgCHnOH In addition to fl-hydroxyethylthioincosaminide, there to L-proline hydrochloride by heating with aqueous hydrochloric acid. The sequence of this latter reaction can be. shown as follows:

i i H 1 i l N CONHNH: N 00011 I101 l H H N-demethyl-7-O-demethylcelesticetin (IV) can be converted to various antibacterially active analogues. Compound (IV) and these analogues can be represented by the following structural formula:

than 20 carbon atoms in the (R) and (S) configuration excepting where X is (R)=OCH R is hydrogen and R is methyl; R is hydrogen or is selected from the group consisting of hydrocarbon carboxylic acid acyl of not more than 18 carbon atoms, or a halo-, nitro-, hydroxy-, amino-, thiocyano-, and lower alkoxy-hydrocarbon carboxylic acid acyl of not more than 18 carbon atoms, inelusive, and

and R is hydrogen, alkyl of from 1 to 20 carbon atoms, inclusive, or hydroxyalkyl of from 2 to 5 carbon atoms, inelusive.

N-demethylcelesticetin (IVa) can be converted to various antibacterially active analogues. These analogues can be represented by the following structural formula:

wherein R is hydrogen or is selected from the group consisting of hydrocarbon carboxylic acid acyl of not more than 18 carbon atoms, or a halo-, nitro-, hydroxy-, amino-, thiocyano-, and lower alkoxy-hydrocarbon carboxylic acid acyl of not more than 18 carbon atoms, inclusive,

ii 0H and R is hydrogen, or alkyl of from 2 to 20 carbon atoms, inclusive, or hydroxyalkyl of from 2 to 5 carbon atoms, inclusive.

N-demethyl-7-O-demethyldesalicetin (V) can be converted to various antibacterially active analo --o (L H CHzCHz O VIIa pound (V) and these analogues can be following. structural formula:

it); E E-N+l H no H K l OH H H L i SCHI'CHIOR] R vnr wherein X, R, and R are as defined above; and R, is hydrogen or is selected from the group consisting of hydrocarbon carboxylic acid acyl of not more than 18 carbon atoms, or a halo-, nitro-, hydroxy-,jamino-, thiocyano-, and loweralkoxy-hydrocarbon carboxylic acid acyl of not more than 18 carbon atoms, inclusive, except that R, is not the salicylic acid moiety when X is (IO-OCH, and R is hydrogen and R is methyl.

N-demethyldesalicetin (Va) can be converted to various antibacterially active analogues. These analogoues can be represented by the following structural formula:

represented by the CHI I CH:o-(BH H VIIIa wherein R and R are as defined above; and R, is hydrogen or is selected from the group consisting of hydrocarbon carboxylic acidacyl of not more than 18 carbon atoms, or a halo-, nitro-, hydroxy-, amino-, thiocyano-, and loweralkoxy-hydrocarbon carboxylic acid acyl of not more than 18 carbon atoms, inclusive; except that R, is not the salicylic acid moiety when R is hydrogen and R is methyl.

5 Hydroxyethylthiolincosaminide (VI) can be converted to various analogues which can be represented by the following structural formula:

K H i on HA wherein X, R, and R are as defined above.

Compounds of formula IX can be converted to antibacterially active compounds by coupling with a pyrrolidinecarboxylic acid as disclosed in US. Pat. 3,514,440. The compounds formed, as well as the process, are disclosed in said patent.

Examples of alkoxy of not more than 20 carbon atoms are methoxy, ethoxy, propoxy, butoxy, pentyloxy, hexyloxy, heptyloxy, octyloxy, nonyloxy, decyloxy, undecyloxy, dodecyloxy, 'tridecyloxy, tetradecyloxy, pentadecyloxy, hexadecyloxy, heptadecyloxy, octadecyloxy, nonadecyloxy, and eicosyloxy and the isomeric forms thereof. Examples of halo are chlorine, bromine and iodine.

Examples of hydrocarbon carboxylic acid acyl of not more than 18 carbon atoms, or a halo-, nitro-, hydroxy-, amino-, thio-,' cyano-, and loweralkoxy-hydrocarbon carboxylic acid acyl of not more than 18 carbon atoms, inelusive, are as disclosed in US. Pat. 3,426,012, column 5, line 64 to column 6, line 47. Examples of alkyl of from 1 to 20 carbon atoms are methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl and eicosyl, and the isomeric forms thereof.

DETAILED DESCRIPTION Chemical and physical properties of N-demethyl-7-O- demethylcelesticetin hydrochloride Elemental analysis:

Calcd. fOI' CggHnNgOgSHClI C, H, N,

5.22; S, 5.97; Cl, 6.53. Calcd. for C H N O S-HCI-H O: C, 47.65; H,

6.31; N, 5.06; S, 5.77; Cl, 6.31. Found: C, 46.78; H, 6.01; N, 5.32; S, 5.34; Cl, 6.13. Molecular weight: The calculated molecular weight for the hydrochloride is 536; N-demethyl-7-O-demethylcelesticetin free base has a molecular weight of 500 (determined by mass spectrometry). Specific rotation:

[a] -=+122 (c., 1, water) =+163 (c., 1, dimethylformamide) Ultraviolet absorption spectra:

In water:

A at 204 mu (a=66.01; e=33,000) A at 238 m (a==17.66; e=8,850) 8 at 303 III/L (a=7.20; e=3,600)

In 0.01 N aqueous NaOH:

A at 241 m (a=11.84; e=5,900) A at 332 III/L (a=8.39; s=4,200)

Infrared absorption spectra: Following is a tabulation of the infrared absorption spectrum wave lengths in mineral oil mull expressed in reciprocal centimeters:

Band frequency (cmr Intensity (oil) 6 Band frequency (cm.- Intensity 852 M 815 M 803 M 760 M 720 M Following is a tabulation of the infrared absorption spectrum wave lengths Where the compound is pressed into a KBr pellet expressed in reciprocal centimeters:

Band frequency (cmr Intensity Infrared band intensities, throughout this disclosure, are indicated as S, M, and W, respectively, and are approximated in terms of the background in the vicinity of the bands. An "S band is of the same order of intensity as the strongest in the spectrum ;M bands are between V3 and as intense as the strongest band; and, W bands are less than as intense as the strongest band. These estimates are made on the basis of a percent transmission scale. SH denotes a shoulder.

Antibacterial activity of N-demethyl-7-O- w mwwmwwm mmmmm demethylcelesticetin Minimum inhibitory Test microorganism: concentration in mcq./ml. Staphylococcus aureus 15.6

Streptococcus hemolyticus 7.8 Streptococcus faecalis 31.2 Escherichia coli 500 Proteus vulgaris 500 Klebsiella pneumoniae 500 Pseudomonas aeruginosa 500 The above antibacterial spectrum was obtained by a tube dilution assay procedure. This procedure was conducted with the medium BHI (Brain Heart Infusion Broth, Difco, Detroit, Mich.). Assay tubes (13 mm. x 100 mm.) were prepared in the customary manner as set out M in Snell, E. E., Vitamin Methods, vol. 1, Academic Press,

7 :Inc., New York (1950), p. 327. Test organisms grown for 18 hours at 37 C. were added to inoculate the test medium. The results were read at 17 hours. Chemical and physical properties of n-demethylcelesticetin hydrochloride Elemental analysis: Elemental analysis showed the presence of C, H, N, S and C1 in the molecule. High resolution mass spectrometry on a trimethylsilyl ether derivative showed molecular ion at 874.3967 mass units (average of 4 determinations). Theoretical for aa 'u a o s is 874.3958 mass units. These results indicate the molecular formula of C I-I N O S for the antibiotic.

'Furthermore the M -CH peak was measured at 859.3724.

Theoretical for C H N O SSi is 859.3723.

Molecular weight: 514 (determined by mass spectrometry).

Specific rotation: [a] =112.5 (c., 1, water). Ultraviolet absorption spectrum (in methanol):

A at 238 mu (a=15.5) A at 304 mu (a=7.2)

Infrared absorption spectra: Following is a tabulation of the infrared absorption spectrum wave lengths in mineral oil mull expressed in reciproca lcentimeters CMFW eral oil mull expressed in reciprocal centimeters:

Band frequency (cm-' 3300 3080 2925 2860 1670 1610 1579 1562 1483 1467 (oil) 1455 (oil) 1375 (oil) 1322 1296 1247 1210 757 p 719 (oil) 701 Following is a tabulation of the infrared absorption spectrum wave lengths where the compound is pressed into a KBr pellet expressed in reciprocal centimeters:

Intensity (oil) (oil) m mzfi zg mwmmmwmmmmmmmwmwmmmm Band frequency (ems- Intensity 3380 S 3260 S 3080 S 29 80 S 2930 M 2820 M 1668 S Band frequency (cmr Antibacterial activity of N-dimethylcelesticetin Minimum inhibitory con- Test microorganism: centration in mcg./ ml. Staphylococcus aureus 15.6 Streptococcus hemolyticus 0.125 Streptococcus faecalis 7.8 Escherichia coli 500 Proteus vulgaris 500 Klebsiella pneumoniae 250 Pseudomonas aeruginosa 500 Diplococcus pneumoniae 0.5

The above test results were obtained using the assay and conditions disclosed above for the antibacterial test of N-demethyl-7-O-demethylcelesticetin.

THE MICROORGAN-ISM The microorganism used for the production of the antibiotics of the subject invention is a mutant of Streptomyces caelestis, NRRL 2418, which has been named Streptomyces caelestis strain 22218a. This mutant is distinguishable from Streptomyces caelestis taxonomically, as shown hereinafter, and by its ability to produce N-demethyl-7-O-demethylcelesticetin and N-demethylcelesticetin. A subculture of the living organism can be obtained from the permanent collection of the Northern Utilization and Research Division, Agricultural Research Service, US. Department of Agriculture, Peoria, 111., U.S.A. Its accession number in this repository is NRRL 5481.

The microorganism of this invention was studied and characterized by Alma Dietz of the Upjohn Research Laboratories.

DESCRIPTION OF THE MIC-ROORGANISM Streptomyces caelestis strain 22218a, UC 5515 Streptomyces caelestis strain 22218a is compared with the parent culture (which is also the type culture) Streptomyces caelestis [De Boer, C., A. Dietz, I. R. Wilkins, C. N. Lewis and G. M. Savage, 1954-1955. Celesticetin- A new, crystalline antibiotic. I. Biologic studies of celesticetin. Antibiotics Annual, New York. Medical Encyclopedia, -Inc., 1955, pp. 831-836], UC 2011, NRRL 2418.

Taxonomic methods: The methods used were those cited in Dietz [Diet-z, A., 1967. Streptomyces stefi'iisburgensis sp. n. J. Bacteriol. 94 :2022-2026] and, in part, those cited in Shirling and Gottlieb [Shirling, 'E. B., and D. Gottlieb, 1966. Methods for characterization of Streptomyces species. Int. J. Syst. Bacteriol. 16:313-340].

Color characteristics: Aerial growth blue (sporulating) for the parent; white with trace blue on some media (non-sporulating to trace sporulating) for the mutant. Parent: melanin-positive; strain: melanin-negative.-Appearance on Ektachrome '[Dietz, A. 1954. Ektachrome 9 10 transparencies as aids in actinomycete classification. Ann. The culture characterized as Streptomyces caelestis N.Y. Acad. Sci. 60:152-154] is given in Table 1. 'Referstrain 222l8a has been so designatedin accordance with ence color characteristics are given in Table 2. The parent Recommendation 8a of the International Code of Noculture may be placed in the White (W), Green (GN), menclature of Bacteria, 1966, edited by the Editorial or Gray (GY) series of Tresner and Backus [Tresner, Board of the Judicial Commission of the International H. D., and E. I. Backus, 1962. System of color wheels Committee on Nomenclature of Bacteria, Intern. I.

for Streptomycete taxonomy. Appl. Microbiol. 11:335- System. Bacteriol. 16: 459-490, in which a number which 338]; the mutant in the White (W) and Yellow (Y) is a laboratory distinguishing mark may be used to desigseries. nate the descendents of a single isolation in pure culture.

Microscopic characteristics: The parent and the mutant The characteristics of Streptomyces caelestis strain have open spiral to spiral sporophores (in the sense of 22218a, NRRL 5481, are given in the following tables:

Pridham et a1. [Pridham, T. G., C. W. Hesseltine, and R. G. Benedict. 1958. A guide for the classification of f li i Appearance of caelems cultures on Ema aocording. to seleqted groups f f Table 2.Reference Color Characteristics of S. caelestis of strams 1n morphological sections. Appl. Microbiol. 5 Cultures 6152-791) :bearmg rectangular. Smcoth Spores the Table 3.-Cultural Characteristics of S. caelestis Cultures sense of Dlfztz P Mathews [Dletz and Mathe.ws' Table 4.lUtilization of Carbon Compounds in the Syn- 1970 Classification 0t Streptomyces spore surf acesinto thetic Medium of Pridham and Gotflieb five groups. Appl. Microbiol. 21Z52:7-533]) W1th ridged Table of Carbon Compounds in the Sym surface structure. The mutant strain has distorted mythetic Medium of Shining and Gottlieb celium with large round bodies when observed by the transmission electron microscope. TABLE 1 Cultural characteristics: See Table 3 Appearance of S. caelestis cultures on Ektachrome l Carbon utilization: See Tables 4 and 5. s. caelestis $606188! Temperature: S. caelestis strain 22218a and the parent 25 medium 5min 22218 NRRL 2418 S. caelestis grow at 18-55 C. Growth is poor at the Bengett's' extremes and optimum at 28-37 C. g gig-ga Antibiotic producing properties: Streptomycas caelestis Czapek's sucro strain 222182. produces 7-O-demethyl-N-demethylceles- 22 gg g ticetin (U-39, 579) and N-demethylcelesticetin (U-40, 30 Maltose-tryptone:

DISCUSSION Pepgoi ie iron:

mutant of Streptomyces caelestz's UC 2011, NRRL 2418, 7 21? Whit 23 is shown to have cultural characteristics distinctly dif- Case iii'starch:

ferent from those of the parent. It does havethe distincz 'ggtggggyfff $2 blue'gmy' tive sporophore and spore characteristics of the parent strain in its sporulating sectors which are celestial blue 4 classification. Ann. N.Y. Acad. Sci. :152-54. (the color of the parents sporulating aerial growth). s=g f TABLE 2 Reference color characteristics of S. caelestis cultures Color harmony manual, 3rd ed., 1948 I l 'N BS circular 553, 1955 h S. caelestis S. caelestia S. caelestia S. caelestis Agar medium strain 22218a NRRL 2418 strain 222182 NRRL 6418 Bennett's:

a white 0 light gray 263 gm white 264 gm light gray. R 20a light ivory, eggshell 2gc bamboo, chamois 89 gm pale yellow 90 gm grayish yellow. P. 2ge covert tan, grieve 94m light olive brown. Czapek's sucrose: h" 3 a w a gm w e. P 2ca light ivory, eggshell 89 gm pale yellow. Maltose tryptone: a wh 0 light gray 263 gm whi 264 gm light y Rt: 20a light ivory, eggshell 31g3 adobelbrlclizvgi, cinnamon 89 pale yellow 77 gm moderate yellowish brown.

rown, g rown.

94m light olive brown m grayish yellowish brown. P 2ge covert glflege 31g beige brown mist brown "{109 gmlight grayish olive 95g moderate olive brown. Yeast extract-mal extract (ISP-2)' S 2ba pearl, shell tint. 0 whi 92 gm yellowish white 364 gniiligllgt gray. R 20a light ivory, eggshell 2ie light mustard tan 89 gm pale yellow i g fi t eggflyfi 941i ht 11 b 9 ii ii ii 'b g 0 ve rown m g 0 ve rown. P zge covert gflege 2E6 covert griege "{104 gm light grayish olive 109 gm light grayish olive, Oatmeal (ISP-3):

S clight gray egray 264 gm hght gray R--. llca cream 1%ec putty 89 gm pale yellow .-{gg i m fi ggy m0 putty {121 m pale yellow green.

' 122 g grayish yellow green. t t%?ii s are s H ht 19d 4 n t 149g pale green.

0 g Y------------------ Y---------------- gm 8 Y---------------- lgomlight bluish gray R 20a light ivory, eggshell 2ec biscuit, ecru, oatmeal, 89 gm pale yellow gm grayish yellow.

san 94 g light olive brown.. 121 m pale yellow green. P 2m covery gray lee light Citron gray ""{112 gm light olive gray" 122 g grayish yellow green, Glggerrogasparagine a whi a a Y a whi 263 gm white 263 gm white. II} 20a light ivory, eggshell..-" 1%ca cream 89 gm pale yellow 89 gm pale yellow. e Jacobson, E., W. C Granville, and C. E. Foss. 1948. Color Harmony Designating Colors and a Dictionary of Color Names. U.S. Dept. 01 Manual, 3d Ed. Container Corporation of America, Chicago. Comm. Circ. 353, Washington, D.C.

b Kelly, K. L., and D. B. Judd. 1955. The ISCC-NBS Method of Now: S=Surtace. R=Reverse. P=Plgment.

l Dietz, A., 1954. Ektachorme tranparencies as aids in actinomycete TAB LE 3 Cultural characteristics of S. caelestf: cultures S. caelesti: strain 22218a S. caelestia, NRRL 2418 Agar medium:

Peptone-iron:

gray. Brown.

Melanin positive.

o Do. Xanthine solubilized Xanthine not soluaround growth. bilized.

Nutrient starch:

S Very slight trace blue- Pale gray.

White. B Cream Cream yellow.

Pale yellow. Yel w. 0 Starch hydrolyze Starch hydrolyzed.

Yeast extractmalt extract:

S Pale blue-white Blue-gray-white.

Yellow Gray-tan. P Pale yellow Tan.

Bennett's:

S White Heavy pale-blue-white. R- Cream- Cream. P Yellow-tan.

Czapeks Pale cream. P

Maltosetryptone:

S White Heavy pale-blue-wbite.

Cream Pale tan. P Do.

Peptone-yeast extract-iron (ISP-B):

S White tinged with blue.. White with trace blue. R Pink-tan Brown. P Light brown.

Gelatin media:

P1 i; Trace brown at surface.

"""""""""""""""""""" Pale yellow-tan brown throughout.

0 No liquefaction No liquefaction. Nutrient:

P Yellow {Trace brown at surface. Yellow-tan throughout. 0- Liquefaction-K N 0 liquefaction.

Broth media: Synthetic nitrate:

S Colorless surface ring with trace white P aerial.

'ir' 'lbris' lift'fii H Growth throughout. 0 growth. Flocculent at base. Nitrate not reduced to Nitrate reduced to nitrite. nitrite.

.. None to very slight trace TABLE 3-Continued S. cnelems strain 22218:: S. caeleatia, NRRL 2418 Utilization of carbon compounds in the synthetic medium of Pridham and Gottlieb S. caelestis S. caelestis strain 222l8a NRRL 2418 Control 1 D-xylose 2 L-arabinose. 3 Rhamn0se- 30 Na succinate Pridham, T. G., and D. Gottlieb. 1948. The Utilization of Carbon Compounds by some Actinomycetalcs as an Aid for Species Determination. J. Bacteriol. 56:107-114.

Nora: +=Good utilization. =Moderate utilization. =Doubtiul utilization. =No utilization.

TABLE 5 Utilization and carbon compounds in the synthetic medium of Shirlinl and Gottlieb 1 S. caeleatia S. caelesti: strain 222188. NRRL 2418 Plain agar (negative control) :i: =1: D-glucose (positive control) L-arabinose Sucrose Y Inositol D-mannit0l. D-iructose..- Rhamnose. Rafiinose-. Cellulose. i :i:

I Shirling, E. B., and D. Gottlieb. 1966. Methods for Characterization of Streptomyces species. Int. J. Syst. Bacteriol. 16:313-340.

No'rn: +=Strong utilization. =Moderate utilization. .-.l==Doubtful utilization. -=No utilization.

The new compounds of the invention are produced when the elaborating organism is grown in an aqueous nutrient medium under submerged aerobic conditions. It is to be understood also that for the preparation of limited amounts surface cultures and bottles can be employed. The organism is grown in a nutrient medium containing a carbon source, for example, an assimilable carbohydrate, and a nitrogen source, for example, an assirnilable nitrogen compound or proteinaceous material. Preferred carbon sources include glucose, brown sugar, sucrose, glycerol, starch, cornstarch, lactose, dextrin, molasses, and the like. Preferred nitrogen sources include cornsteep liquor, yeast, autolyzed brewer's yeast 13 with milk solids, soybean meal, cottonseed meal, cornmeal, milk solids, pancreatic digest of casein, distillers solids, animal peptone liquors, meat and bone scraps, and the like. Combinations of these carbon and nitrogen sources can be used advantageously. Trace metals, for example, zinc, magnesium, manganese, cobalt, iron, and. the like, need not be added to the fermentation media since tap water and unpurified ingredients are used as components of the medium.

Production of the compounds of the invention can be effected at any temperature conducive to satisfactory growth of the microorganism, for example, between about 18 and 40 C., and preferably between 20 and 32 C. Ordinarily, optimum production of the compounds is obtained in about 2 to days. The medium normally remains basic during the fermentation. The final pH is dependent, in part, on the buffers present, if any, and in part on the initial pH of the culture medium.

When growth is carried out in large vessels and tanks, it is preferable to use the vegetative form, rather than the spore form, of the microorganism for inoculation to avoid a pronounced lag in the production of the new compounds and the attendant inefficient utilization of the equipment. Accordingly, it is desirable to produce a vegetative inoculum in a nutrient broth culture by inoculating this broth culture with an aliquot from a soil or a slant culture. When a young, active vegetative inoculum has thus been secured, it is transferred aseptically to large vessels or tanks. The medium in which the vegetative inoculum is produced can be the same as, or different from, that utilized for the production of the new compounds, as long as it is such that a good growth of the microorganism is obtained.

The new compounds of the invention, N-demethyl-7-O- demethylcelesticetin (IV) and N-demethylcelesticetin (IVa), exist either in the non-protonated (free base) form or the protonated (salt) form depending on the pH of the environment. They form stable protonates (acid addition salts) by neutralization of the free base with an acid or by metathesis between the protonated form of the antibiotic and the anion of an acid. Suitable acids for this purpose include hydrochloric, sulfuric, phosphoric, acetic, succinic, citric, lactic, maleic, fumaric, pamoic, cholic, palmitic, mucic, camphoric, glutaric, glycolic, phthalic, tartaric, lauric, stearic, salicylic, S-phenylsalicylic, S-phenylsalicylic, 3-methylglutaric, orthosulfobenzoic, cyclohexanesulfamic, cyclopentanepropionic, 1,2 cyclohexanedicarboxylic, 4-cyclohexenecarboxylic, octadecenylsuccinic, octenylsuccinic, methanesulfonic, benzenesulfonic, helianthic, Reineckes, azobenzenesulfonic, octadecylsulfuric, picric, and the like acids. These acid addition salts are useful in upgrading the free base.

Because of the presence of the salicylic acid moiety, N-demethyl-7-O de-methylcelesticetin (IV) and =N-demethylcelesticetin (IVa) form salts with alkali metals and alkaline earth metals by procedures well known in the art. Salts which can be made are the sodium, potassium, calcium, lithium, and the like. These salts have the uses disclosed above for the acid addition salts.

N-demethyl-7-O-demethylcelesticetin (IV) and N-demethylcelesticetin ('IVa) are soluble in lower alcohols of from 1 to 4 carbon atoms, inclusive, and ketones; they are less soluble in water and chlorinated hydrocarbon solvents; and essentially insoluble in ether and saturated hydrocarbon solvents.

A variety of procedures can be employed in the isolation and purification of N-demethyl 7 O demethylcelesticetin (IV) and N-demethylcelesticetin (IVa), for example, solvent extraction, partition chromatography, silica gel chromatography, liquid-liquid distribution in a Craig apparatus, and crystallization from solvents.

In a preferred recovery process, N-demethyl-7-O-demethylcelesticetin (IV) and N-demethylcelesticetin (IVa) are recovered from the culture medium by separation of the mycelia and undissolved solids by conventional means,

such as by filtration or centrifugation. The antibiotics are then removed from the filtered or centrifuged broth by resin. sorption on a resin comprising a non-ionic macro porous copolymer of styrene crosslinked with divinylbenzene. Suitable resins are Amberlite XA-D-l and XAD-Z disclosed in US. Pat. 3,515,717. The resin is eluted with an organic or aqueous organic solvent in which the sorbed antibiotics'are soluble. Ninety-five percent aqueous methanol is the preferred solvent for elution. The antibiotic mixture in the methanol eluates is purified by concentrating the eluates to dryness and then subjecting them to counter double current distribution using l-butanol-water (1:1 v./v.) as the solvent system. Fractions containing N-demethylJ-O-demethylcelesticetin are concentrated to dryness and the antibiotic crystallizes by triturating with methanol.

Fractions containing N-demethylcelesticetin are subjected to countercurrent distribution using a solvent system consisting of l-butanol-water (1:1 v./v.). The N-demethyl-7-O-demethylcelesticetin which is present in small amounts in collected fractions which contain predominantly N-demethylcelesticetin is isolated by use of silica gel chromatography. This purification step affords a facile separation of the antibiotics. Fractions from the silica gel chromatography column which contain only N-demethylcelesticetin are concentrated to dryness; the resulting residue is dissolved in methanolic hydrogen chloride, and this solution is mixed with ether. The resulting precipitated N-demethylcelesticetin hydrochloride is isolated by filtration and dried.

The presence of N-demethyl-7-O-demethylcelesticetin (IV) and N-demethylcelesticetin (Na) in the fermentation beer and various recovery liquids is determined by activity against the microorganism S. lutea. This is a standard microbiological disc plate assay using 12.5 mm. discs. Thin layer chromatography (TLC) using silica gel G and a solvent mixture consisting of chloroform-methanol (6:1 v./v.) is used to identify the presence of either or both of the antibiotics in a sample showing activity against S. lutea, as described above. On this chromatographic system, N-deme thyl-7-O demethylcelesticetin ('IV) has an R of 0.05 and N-demethylcelesticetin (IV a) has an Rf of 0.15.

Alkaline hydrolysis of N-demethyl-7-O-demethylcelesticetin (IV) and N-demethylcelesticetin (VIa) with a suitable base, for example, sodium hydroxide, for 16 hours at room temperature results in the cleavage of the ester group and the formation of N-demethyl-7-O-demethyldesalicetin (V) and-N-demethyldesalicetin (Va), respectively. Other bases, for example, aqueous ammonium hydroxide and organic amines can be used instead of sodium hydroxide. The reaction can be carried out in solvents like lower alcohols (methanol, ethanol) by refluxing in the presence of base. Duration of the treatment depends on the strength of the base and the temperature used.

Salicylic acid, which is formed as a by-product during the above reaction, can be removed by extraction with ether at pH 3.0.

Hydrazinolysis of N-demethyl-7-O-demethylcelesticetin (IV) with hydrazine hydrate at reflux for about 23 hours affords proline hydrazide which can be transformed to proline hydrochloride by heating with aqueous hydrochloric acid. The second product of the above hydrazinolysis reaction is fl-hydroxyethylthiolincosaminide (p-HETL) (VI).

N-demethyl-7-O-demethyldesalicetin (V) and N-demethyldesalicetin (Va) exist either in the non-protonated (free base) form or the protonated (salt) form depending on the pH of the environment. Acid addition salts of these compounds can be made as disclosed for the N-demethyl-7-O-demethylcelesticetin (IV) and N-demethylcelesticetin (IVa).

N-demethyl-7-O-demethylcelesticetin (IV) and N-demethylcelesticetin (IVa) and their salts are active against Staphylococcus aureus and Streptococcus faecalis and can be used to disinfect washed and stacked food utensils contaminated with these bacteria; they can also be used as disinfectants on various dental and medical equipment contaminated with Staphylococcus aureus. Further, since these antibiotics are active against Streptococcus hemolyticus, they can be used to disinfect instruments, utensils or surfaces where the inactivation of this organism is desirable.

N-demethyl-7-O-demethyldesalicetin (V) and N-demethyldesalicetin (Va), and their salts, are active against Staphylococcus aureus and can be used for the same purposes given for N-demethyl-7-O-demethylcelesticetin (IV) and N-demethylcelesticetin (VIa).

N-demethyl-7-O-demethylcelesticetin (IV) and N-demethylcelesticetin (IVa) can be converted to various antibacterially active analogues as represented by structural formulae VII, VIIa, VIII, and VIIIa. In the following discussion, in cases where acylation, phosphorylation, or 7-epimerization via 7-oxidation ofcompounds, IV, IVa, V, or Va are involved, the compound being reacted should be suitably N'protected according to the art, for example as the N-carbobenzoxy derivtaive. The carbobenzoxy group is removed after reaction or at a suitable later time by hydrogenolysis, according to the art. For example, N-demethyl-7-epi-7-O-demethylcelesticetin can be prepared by the procedure disclosed in U.S. 3,514,440, column 2, line 44 to column 3, line 10; N-demethyl-7 (R)- and 7(S)-halo-7-demethoxycelesticetin can be prepared by the methods disclosed in U.S. 3,496,163, using the modified Rydon procedure as given in Example 6 of said patent; N demethyl-7(R)-O-alkyl-7-O-demethylcelesticetin can be prepared by esterification of N-demethyl- 7 (R)-a1kyl-7-O-demethyldesalicetin at the primary hydroxyl with salicyloyl chloride as disclosed in U.S. 2,851,463, especially column 2, lines 70 et seq.; N-demethyl-7(S)-O-alkyl-7-O-demethylcelesticetin can be prepared from N-demethyl-7(S)-O-alkyl-7-O-demethyldesalicetin by the procedure disclosed above for the preparation of the 7(R) isomer; 2-Ophosphates can be prepared by the procedures disclosed in U.S. 2,487,068; Z-O acylates can be prepared by the acylation procedures disclosed in U.S. 3,326,891 and U.S. 3,426,012; and N-alkyl compounds of formulae VII and VIIa can be prepared by procedures disclosed in U.S. 3,380,992 with the provision that the starting compound of formula V in U.S. 3,380,992 is to be the present compound of formula IV or IVa in the subject invention; i.e. wherein Y of formula V in U.S. 3,380,992 is a-SCHaCHzO 11 R is CH;, and R, R and R are H for IV and R and R are -CH R and R are H for IVa. The most pertinent portions of U.S. 3,380,992 are column 2, lines 17 et seq., column 13, lines 43-69, and Example 1, parts F-2 to F-4 and G-l, column 19, line 74 to column 20, line 52.

N-hydroxyalkyl compounds for formulae VII and VIIa can be prepared by reacting the compound of formula IV or IVa, advantageously as the hydrochloride acid addition salt, with ethylene oxide, propylene oxide, butylene oxide or pentylene oxide, preferably in a pressure vessel in which the reaction proceeds as follows as illustrated with ethylene oxide:

H 1's N The reaction mixture after cooling is worked up in the usual manner using such procedures as distillation, solvent extraction, crystallization and the like.

The proportions are not critical but an excess of alkylene oxide is desirable. An excess of from 2 to 100 times the stoichiometric amount is suitable. The temperature also is not critical. At temperatures below about 35 C., however, the reaction is undesirably slow and ordinarily it will not be necessary or desirable to exceed about 200 C. The reaction proceeds well at C.

Advantageously the reaction is carried out in an inert mutual solvent for the reactants. Suitable such solvents include methyl alcohol, ethyl alcohol, propyl alcohol, benzene, toluene, cyclohexane, and tetrahydrofuran.

Alternatively the alkylation can be effected by a reductive alkylation such as described in U.S. Pat. 3,496,- 163, Part 614, by substituting the formaldehyde (formalin) by hydroxyacetaldehyde, 3-hydroxypropionaldehyde, 4-hydroxybutyraldehyde or S-hydroxypentaldehyde. Salts of the compounds of structural formulae VII and VIIa can be made as disclosed for compounds IV and IVa. These salts are useful in the same manner as the non-protonated compounds, and further, they can be used to upgrade the non-protonated compounds by procedures well known in the art.

N-demethyl-7-O-demethyldesalicetin (V) and N-demethyldesalicetin (Va) can be converted to various antibacterially active analogues as represented by structural formulae VIII and VIIIa. For example, N-demethyl-7- epi-7-O-demethyldesalicetin can be prepared by hydrolysis of the B-O-salicylyl acyl of N-demethyl-7-epi-7-0-demethylcelesticetin by methods disclosed in U.S. 2,851,- 463, especially column 1, line 27 to column 2, line 56 [N-demethyl-7-epi-7-O-demethylcelesticetin can be prepared by methods disclosed in U.S. 3,514,440, as noted above]; p-acylates of N-demethyl-7-epi-7-O-demethyldesalicetin and N-demethyldesalicetin can be prepared by the procedures disclosed in U.S. 2,851,463, particularly those disclosed in column 2, line 56 to column 3, line 16; N-demethyl-7(R)- and -7(S)-halo-7-demethoxydesalicetin can be prepared by hydrolysis of N-demethyl- 7(R)- and -7(S)-halo-7 demethoxycelesticetin by methods disclosed in U.S. 2,851,463, especially column 1, line 27 to column 2, line 56, except that the process should be carried out at room temperature, at a pH of 8-10, preferably about pH 10, so as to selectively remove the salicyloyl and not hydrolyze off the 7-halo group [N-dimethyl-7(R)- and -7(S)-halo-7-demethoxycelesticetin can be prepared by methods disclosed in U.S. 3,496,163, as noted above]; N-demethyl-7(R)-O- alkyl-7-O-demethyldesalicetin can be prepared by reacting N-demethyl-7 (R)-O-alkyl-HETL [this compound can be prepared by substituting HE'IL (VI) for the compound methyl thiolincosaminide (MTL) in the procedures disclosed in U.S. 3,574,187] with ethyl chloroformate according to methods known in the art to give the N- demethyl-7(R)-0-alkyl-HETL p-O-ethylcarbonate ester which then can be N-acylated with N-carbobenzoxy-L- proline by the procedures disclosed in U.S. 3,380,992, especially Example 6the ethyl carbonate ester protective group can be removed by treatment with a mild base, e.g. NaHCO N-demethyl-7(S)-O-alkyl-7-O-demethyldesalicetin can be prepared from N-demethyl- 7(S)-O-alkyl-7-0-demethyl-HETL according to the procedure given above for the 7(R) isomer; 2-O-phosphates can be prepared by first preparing the ,B-O-(alkyl carbonate)-ester of N-demethyl-7 0-demethyldesalicetin (V) or N-demethyldesalicetin (Va) by reaction of the desalicetin compound with an alkyl chloroformate using procedures known in the art, and then phosphorylating the reaction product according to the procedures disclosed in U.S. 3,487,068-the alkyl carbonate protective group can be removed by treatment of the phosphorylated product with mild base, e.g. NaHCO fl-O-acylates can be prepared by the procedures disclosed in U.S. 2,851,- 463, especially column 2, lines 70 et seq. using one mole of acylating agent per mole of desalicetin compound; 2- O-acylates can be prepared by the acylation procedures disclosed in U.S. 3,326,891 and U.S. 3,426,012; N-a'lkyl and N-hydroxyalkyl compounds 9i formulae VIII and 17 VIIIa can be prepared as described above for similar derivatives of formulae VII and VHa.

' Acid addition salts of the compounds of structural formulae VIII and VIIIa can be made as disclosed for compounds IV and Na. These salts are useful in the same manner as the non-protonated compounds, and, further, they can be used to upgrade the nonprotonated compounds by procedures well known in the art.

The following examples are illustrative of the process and products of the present invention, but are not to be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted.

EXAMPLE 1 N-demethyl-7-O-demethylcelesticetin (IV) Gm./l. Glucose monohydrate 25 Pharmamedia 1 25 Tap water, q.s. 1 liter.

1 Pharmamedia is an industrial grade of cottonseed flour produced by Trader's Oil Co., Fort Worth, Tex.

The presterilization pH of the seed medium is 7.2. The seed is grown for three days at 28 C. on a Gump rotary shaker operating at 250 r.p.m.

The fermentation proper is carried out in the medium of the following composition:

I Gm./l. Glucose monohydrate 25 Wilsons Peptone Liquor No. 159 1 15 Yeast extract 2.5

Tap water, q.s. 1 liter.

Wilson's Peptone Liquor No. 159 is a preparation of enzymatically hydrolyzed proteins of animal origin.

The medium is adjusted to pH 7.2-7.4 with 50% sodium hydroxide, distributed in 100 ml. volumes/500 ml. Erlenmeyer flasks and sterilized at 120 C. for thirty (30) minutes. The inoculated flasks (5% inoculum) are incubated at 28 C. on a Gump rotary shaker operating at 250 r.p.m. Optimum production of N-demethyl-7-O- demethylcelesticetin (IV) and N demethylcelesticetin (IVa) is obtained after about four days of fermentation. The titer of these antibiotics in the fermentation beer is determined by the activity of the antibiotics against the microorganism S. lutea by a standard microbiological disc plate assay using 12.5 mm. discs.

A scaled-up fermentation process is as follows:

Shake flask pre-seed medium: Gm./l. Glucose monohydrate 10 Wilsons Peptone Liquor No. 159 10 Cornsteep liquor 10 Pharmamedia 2 Tap water, q.s. 1 liter.

The medium is adjusted to pH 7.2 with aqueous sodium hydroxide, sterilized in a series of 500-ml. Erlenmeyer flasks each containing 100 ml. of seed medium. The flasks are inoculated from slants with Streptomyces caelestis strain 22218a, NRRL 5481, and the seed flasks are then grown for three days at 28 C. on a Gump rotary shaker operating at 250 r.p.m. Three shake flasks (300 ml.) of the pre-seed inoculum, described above, are used to inoculate a 300-liter seed tank containing 250 liters of the seed medium described above for the seed flasks with the addition of Ucon (a polyalkylene glycol synthetic defoaming fluid supplied by Union Carbide Chemical Co.) as an antifoaming agent. The seed tank is maintained at a temperature of 28 C. for 48 hours Gm./l. Glucose monohydrate 15 Wilsons Peptone Liquor No. 159 15 Yeast extract l Ucon is added as an antifoaming agent. The fermentation tank is inoculated at the rate of 5% seed and maintained at 28 C. for four days with an air flow of s.c.f.m. at 10 p.s.ig. and agitation at a rate of 166 r.p.m.

Part B.-Recovery.-Whole fermentation beer (approximately 4400 liters), obtained as described above, is filtered using diatomaceous earth as a filter aid. The filter cake is washed with water. The wash is combined with the clear beer and the combined solution is passed through 150 liters of Amberlite XAD-Z resin at a flow rate of 150 liters per hour. The spent beer is discarded. The column is washed with water (75 liters) and then eluted with 1100 liters of aqueous methanol. The methanolic eluates are collected in two pools: Pool I (430 l.) is concentrated to dryness; yield, 1275 g. Pool II (970 l.) is also concentrated to dryness; yield 343 g.

Part C.Isolation of N-demethyl 7 O demethylcelesticetin hydrochloride by counter double current distribution: A portion of Pool I, described above, is subjected to purification by counter double current distribution using a solvent system consisting of l-butanol-water (1:1) to obtain crystalline N-demethyl 7-O-demethylcelesticetin hydrochloride. 50 g. of the material obtained from Pool I is dissolved in both phases (200 ml. of each phase) of the above-noted solvent system and the pH is adjusted to 3.5 with 2 N aqueous hydrochloric acid. The solution is put in tubes 12-20 at the point where the lower phase enters the countercurrent distribution machine. The following transfers are run:

(1) 30 transfers without collecting fractions. (2) 32 transfers with collection of the upper phase only. (3) 50 transfers collecting both upper and lower phases.

The distribution is analyzed by determination of bioactivity of selected fractions against S. lutea.

Fraction N0.:

Lower collector: Zone (mm.) of inhibition 19 TABLEContinued Fraction No.:

Upper machine: Zone (mm.) of inhibition Selected fractions are pooled. Each pool is concentrated to dryness in vacuo to give the following preparations respectively.

Pool AI, lower collector 3050; lower machine 50-46. P001 BI, lower machine 37-0. Pool CI, upper collector -0. Pool AI contains traces of bio-activity and is discarded. Pool BI is dissolved in a minimum amount of methanol and the solutions are mixed with 200 ml. of ether. The resulting precipitated material is collected by filtration and dried; yield, 2.13 g., labelled BI-l.

Pool CI is triturated with 200 ml. of ether and the insoluble material (1.45 g.) is labelled CI-l.

Following the above procedures, five additional counter double current distribution runs are performed using 50 g. of Pool I starting material each time. From Pool BII (lower machine (40-0)) there are obtained five preparations which are combined and triturated with 150 ml. of absolute methanol. The resulting insoluble crystalline material is analyzed and the results show that this material (700 mg.) is pure N-demethyl-7-0-demethylcelesticetin hydrochloride (IV).

EXAMPLE 2 N-demethylcelesticetin (We) The filtrate from the trituration with methanol of the five preparations obtained from Pool BII, as disclosed in Example 1, is added to two liters of ether. The resulting precipitated material is isolated by filtration and dried; yield, 10.63 g., labelled BII-l. This material is combined with preparation labelled BI-1, obtained as disclosed in Example 1, to give 12.7 g. of material containing N-demethylcelesticetin (Na), and labelled -BII-2. This material is then subjected to counter current distribution using the solvent system l-butanol-water (1:1 v./v.). The starting material (12.7 g. of preparation BII2) is dissolved in both phases of the above-noted solvent system and added into five tubes of a SOO-tube all glass counter current distribution apparatus. The distribution is analyzed for antibiotic content by using Sarcina lutea as the test organism.

After 1500 transfers, tubes 340-400 are combined and the solution concentrated to dryness. The residue is dissolved in 50 ml. of methanol and this solution is mixed with one liter of ether to give a precipitate; yield, 1.5 g. labelled BII-3. This material is found by TLC chromatography (silica gel G, chloroform-methanol 6:1 v./v.)

to be a mixture of N-demethylcelesticetin (Na) and N- demethyl-7-O-demethylcelesticetin (IV). These two antibiotics are separated by silica gel chromatography as fol lows: The silica gel chromatography column is prepared from 250 g. of silica gel G (Merck-Darmstadt ART. 7734) packed in the solvent system consisting of chloroform-methanol (6:1 v./v.). The starting material (1.3 g. of BIL-3) is dissolved in the solvent and the solution is mixed with 20 g. of silica gel. The mixture is concentrated to dryness and the obtained powder is added on the top of the column bed. The column is eluted with the above solvent system. Fractions (20 ml. each) are analyzed by bio-activity against Sarcina lutea and ultraviolet determinations. Results follow.

Fraction Zone (8. number lutea, mm.) UV (X mlx- 0D) Noudeseriptive UV.

Traces Do.

EXAMPLE 3 N-demethyl-7-O-demethyldesalicetin (V) Upon the treatment of N-demethyl-7-O-demethylcelesticetin (IV) with 50% aqueous NaOH for about 16 hours at room temperature, there is produced N-demethyl- 7-O-demethyldesalicetin (V). Salicylic acid which is formed as a byproduct during the reaction can be removed by extraction with ether at pH 3.0.

EXAMPLE 4 N-demethyldesalicetin (Va) Upon the treatment of N-demethylcelesticetin (IVa) with 50% aqueous NaOH for about 16- hours at room temperature, there is produced N-dernethyldesalicetin (Va). Salicyclic acid which is formed as a by-product during the reaction can be removed by extraction with ether at pH 3.0.

EXAMPLE 5 B-Hydroxyethylthiol incosaminide (p-HETL) (VI) Upon hydrazinolysis of N-demethyl-7-O-demethylcelesticetin (IV) with hydrazine hydrate at reflux for about 23 hours, where is produced fl-HETL, proline hydrazide and salicylic acid hydrazide. ,s-HETL is recovered from the reaction mixture by counter current distribution in the solvent system consisting of equal volumes of l-butanol and water. This procedure separates p-HETL from proline hydrazide and salicylic acid hydrazide.

What is claimed is:

1. A compound having the structural formula:

I OH:

wherein X is OH in the (R) and (S) configuration; halo, wherein halo is chlorine, bromine, or iodine, in the (R) and (S) configuration; alkoxy of not more than 20 carbon atoms in the (R) and (S) configuration; R is selected from the group consisting of hydrocarbon carboxylic acid acyl of not more than 18 carbon atoms, or a halo-, nitro-, hydroxy-, amino-, thiocyano-, and loweralkoxy-hydrocarbon carboxyic acid acyl of not more than 18 carbon atoms, inclusive, and

ii H

and R is hydrogen, or alkyl of from 2 to 20 carbon atoms,

inclusive, or hydroxyalkyl of from 2 to 5 carbon atoms, inclusive; and salts thereof.

2. A compound having the structural formula:

CHaO- H ii HO o H J[ cmomoiiQ n vm 22 wherein R is selected from the group consisting of hydrocarbon carboxylic acid acyl of not more than 18 carbon atoms, or a halo-, nitro-, hydroxy-, amino-, thiocyano-, and loweralkoxy-hydrocarbon carboxylic acid acyl of not more than 18 carbon atoms, inclusive,

ii oH and R is hydrogen, or alkyl of from 2 to 20 carbon atoms, inclusive, or hydroxyalkyl of from 2 to 5 carbon atoms, inclusive; and salts thereof.

3. A compound having the structural formula:

ii HO H H OH H CHzCHzORa R VIII wherein X, R, and R are as defined in claim 1 and R is selected from the group consisting of hydrogen, hydrocarbon carboxylic acid acyl of not more than 18 carbon atoms, or a halo-, nitro-, hydroxy-, amino-, thiocyano-,

and loweralkoxy-hydrocarbon carboxylic acid acyl of not more than 18 carbon atoms, inclusive; and acid addition salts thereof.

4. A compound having the structural formula:

I CH3 arm-on H 6 C-N H ii HO H OH H)i (L scrnornon, H R VIIIa wherein R and R are as defined in claim 1; and R is selected from the group consisting of hydrogen hydrocarbon carboxylic acid acyl of not more than 18 carbon atoms, or a halo-, nitro-, hydroxy-, amino-, thiocyano-, and loweralkoxy-hydrocarbon carboxylic acid acyl of not more than 18 carbon atoms, inclusive; and acid addition salts thereof.

References Cited UNITED STATES PATENTS I v UNITED sTATEs PATENT OFFICE CERTIFICATE OF CORRECTION Patent No. 5,812,096 Dated M jl, 1974 Inventor-(a) A Argoudel J -H CO8 [15, and O. K. Sebek It is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below:

Column 5, l ine 1, for "B-hydroxyethylthioin'cosaminide" read B-hydroxyethylthiolincosaminide line 52, for "R;=OCH read 2R)-OCH Column 4, line 20, for

R -OCH read R)-0CH Column 6, l ine 62, for "mcq." read mcg. Column 7, l ines 27-28, for "reciproca .lcentlmeters- CMFW eral oi l mul l expressed in reciprocal centimeters read reciprocal centimeters Column 10, l ine 58, for "E ktachorme" read Ektach rome Col umn 5, table 2, for "6418" read 2418 Column 12', table 5, line 2, For "A ar medium" read Broth media Column 12, table 5, line 4 for "and" read of line 62, for read Column 15, l ine 22, for "derivtaive" read derivative l ine 55, for -"-7(R)al kyl read 7(R) O-al kyl l ine 40, y for "2,487,068 read 5,487,068 Column 21, l ine 9, for "'Salicycl ic" read Sal icyl ic line 17, For "where" read there l ine 46, For "carboxyic" read carboxyl ic Column 22, l ine 45-46, for hydrogen hydrocarbon" read hydrogen, hydrocarbon Signed and sealed this 1st day of October 1974.,

(SEAL) Attest:

' McCOY M. GIBSON JR. C. MARSHALL DANN Attesting Officer Commissioner of Patents USCOMM-DC 60376-P60 i U.S. GOVIHNMENT PRINTING OFFICE "II 0-366-334 FORM PO-lOSO (10-53) 

